Haemopoietin receptor and genetic sequences encoding same

ABSTRACT

The present invention is directed to a novel haemopoietin receptor or a derivative thereof and to genetic sequences encoding same. The receptor molecule and its derivatives and the genetic sequences encoding same of the present invention are useful in the development of a wide range of agonists, antagonists, therapeutics and diagnostic reagents based on ligand interaction with its receptor. The present invention particularly relates to a receptor for leptin.

BACKGROUND OF THE INVENTION

The preferred haemopoietin receptor of the present invention is referredto herein as “NR2”. The NR2 receptor interacts with leptin and isreferred to as a “leptin receptor”. The terms “haemopoietin receptor”,“NR2 and “leptin receptor” are used in interchangeably throughout thesubject specification. The species from which a particular NR2 isdesired is given in single letter abbreviation in lower case before NR2.For example, murine NR2 is “mNR2” and human NR2 is “hNR2”. A recombinantform may have the prefix “r”.

The rapidly increasing sophistication of recombinant DNA techniques isgreatly facilitating research into the medical and allied health fields.Cytokine research is of particular importance, especially as thesemolecules regulate the proliferation, differentiation and function of awide variety of cells. Administration of recombinant cytokines orregulating cytokine function and/or synthesis is becoming increasinglythe focus of medical research into the treatment of a range of diseaseconditions.

Despite the discovery of a range of cytokines and other secretedregulators of cell function, comparatively few cytokines are directlyused or targeted in therapeutic regimums. One reason for this is thepleiotropic nature of many cytokines. For example, interleukin (IL)-11is a functionally pleiotropic molecule (1,2), initially characterized byits ability to stimulate proliferation of the IL-6-dependentplasmacytoma cell line, T11 65 (3). Other biological actions of IL-11include induction of multipotential haemopoietin progenitor cellproliferation (4,5,6), enhancement of megakaryocyte and plateletformation (7,8,9,10), simulation of acute phase protein synthesis (11)and inhibition of adipocyte lipoprotein lipase activity (12, 13). Thediverse and pleiotropic function of IL-11 and other haemopoietincytokines makes these molecules an important group to study, especiallyat the level of interaction of the cytokines with their receptors.Manipulation and control of cytokine receptors and of cytokine-receptorinteraction is potentially very important in many therapeuticsituations, especially where the target cytokine is functionallypleiotropic and it is desired to block certain functions of a targetcytokine but not all functions.

Another important aspect of cytokine receptors is in the search for newcytokines. In this regard, the inventors have used a procedure forcloning haemopoietin receptors without prior knowledge of their ligands.Identification of receptors then provides a screening procedure forpotentially new cytokines and for previously characterised cytokines. Inaddition, identification of new haemopoietin receptors allows forselective blocking of pleiotropic cytokine function.

In accordance with the present invention, the inventors identified anovel haempoietin receptor which interacts with leptin, a hormone whichregulates adipose tissue mass.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a novel haemopoietin receptor or aderivative thereof and to genetic sequences encoding same. The receptormolecule and its derivatives and the genetic sequences encoding same ofthe present invention are useful in the development of a wide range ofagonists, antagonists, therapeutics and diagnostic reagents based onligand interaction with its receptor. The present invention particularlyrelates to a receptor for leptin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic resentation showing size of NR2 cDNA clonesisolated and schematic structure of the predicted NR2 protein.

FIGS. 2A to 2K are a representation of the nucleotide sequence andcorresponding amino acid sequence of the haemopoietin receptor.

FIG. 3 is a representation of a FACS analysis of NR2 expression by BA/F₃cells.

FIG. 4 is a photographic representation of a silver-stained gel of NR2expression by BA/F₃ cells.

FIGS. 5A and 5B are a graphical representation showing specific bindingof ¹²⁵I human leptin to Ba/F₃ cells stably transfected to express hNR2on their cell surface.

(a) Saturation binding curve for ¹²⁵I h leptin binding to Ba/F3/hNR2cells at 23° C.

(b) Scatchard transformation of the data in (a). The slope of the curveindicates an equilibrium dissociation constant (K_(D)) of 120 pM.

FIG. 6 is a graphical representation showing specific binding of ¹²⁵Ihuman leptin to COS-7 cells transiently transfected to express hNR2 ontheir cell surface (a) or to purified soluble human NR2 (b). Saturationbinding curves at 23° C. are shown.

FIG. 7 is a photographic representation showing cross speciesconservation of the NR2 gene. Southern blot of genomic DNA probed with aspecific cDNA probe for NR2.

FIG. 8 is a diagrammatic representation of the NR2 locus. A map of theNR2 locus, showing positioning of the clones isolated from genomiclibraries. The results of the restriction enzyme mapping using NcoI andthe positioning of the exons of these fragments are also shown.

FIG. 9 is a photographic representation showing expession of leptinreceptor (NR2) in murine tissues.

DETAILED DESCRIPTION OF THE INVENTION

Bibliographic details of the publications numerically referred to inthis specification are collected at the end of the description. SequenceIdentity Numbers (SEQ ID NOs.) for the nucleotide and amino acidsequences referred to in the specification are defined following thebibliography.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated integer or group of integers but not the exclusion of anyother integer or group of integers.

One aspect of the present invention is directed to an isolated nucleicacid molecule comprising a sequence of nucleotides encoding orcomplementary to a sequence encoding a haemopoietin receptor or aderivative thereof wherein said sequence of nucleotides or acomplemantary form thereof is capable of hybridising under mediumstringent conditions to the oligonucleotide:

5′-(A/G)CTCCA(A/G)TC(A/G)CTCCA-3′ [SEQ ID NO: 1].

In a preferred embodiment, the nucleic acid molecule comprises anucleotide sequence or a complementary form thereof which hybridisesunder medium stringent conditions to the oligonucleotides:

5′-(A/G)CTCCA(A/G)TC(A/G)CTCCA-3′ [SEQ ID NO:1]

5′-ACTAGCAGGGATGTAGCTGAG-3′ [SEQ ID NO:4]

5′-CTGCTCCTATGATACCT-3′[SEQ ID NO:6]

5′-CCTCTTCCATCTTATTGCTTGG-3′ [SEQ ID NO:7]

 5′-ATCGGTCGTGACATACAAGG-3′[SEQ ID NO:8].

In an even more preferred embodiment, the nucleic acid moleculecomprises a nucleotide sequence or a complementary form thereof whichhybridises under medium stringent conditions to one or more of thefollowing oligonucleotides:

5′-(A/G)CTCCA(A/G)TC(A/G)CTCCA-3′ [SEQ ID NO: 1].

5′-ACTAGCAGGGATGTAGCTGAG-3′ [SEQ ID NO:4]

5′-CTCAGCTACATCCCTGCTAGT-3′ [SEQ ID NO:5]

5′-CTGCTCCTATGATACCT-3′ (SEQ ED NO:6]

5′-CCTCTTCCATCTTATTGCTTGG-3′ [SEQ ID NO:7]

5′-ATCGGTCGTGACATACAAGG-3′[SEQ ID NO:8]

5′-AGCTAAGCTTTCTAGATATCCAATTACTCCTTGGAGA-3′[SEQ ID NO:9]

5′-AGCTTCTAGATCAAACTTTTATATCCATGACAAC-3′[SEQ ID NO:10]

5′-AGCTTCTAGATCAAACTTTTATATCCATGACAAC-3′[SEQ ID NO: 11].

In a still more preferred embodiment, the nucleic acid moleculecomprises a nucleotide sequece or complementary form thereof which iscapable of hybridising separately under medium stringent conditions toeach of oligonucleotide SEQ ED NO:1 and SEQ ID NO:4 to SEQ ID NO:11.

In a most preferred embodiment, the present invention provides a nucleicacid molecule comprising a sequence of nucleotides or a complementaryform thereof substantially as set forth in FIG. 2 [SEQ ID NO: 12] or asequence of nucleotides capable of hybridising to all or part thereofunder medium stringent conditions.

Accordingly, a preferred embodiment of the present invention is alsodirected to a nucleic acid molecule encoding a haemopoietin receptor ora derivative thereof and comprising a nucleotide sequence as set forthin SEQ ID NO:12 or is capable of hybridising to all or part thereofunder medium stringent conditions.

The haemopoietin receptor of the present invention is referred to hereinas “NR2”. In accordance with the present invention, NR2 is capable ofinteracting with leptin and, hence, is also referred to as a “leptinreceptor”.

The term “derivative” includes any or all parts, fragments, portions,homologues or analogues to the nucleotide sequence set forth in SEQ IDNO:12 as well as hybrid molecules between the NR2 receptor and otherreceptors or other molecules. Derivatives include single or multiplenucleotide substitutions, deletions and/or additions to the nucleotidesequence set forth in SEQ ID NO:12.

Another aspect of the present invention contemplates a recombinanthaemopoietin receptor encoded by the nucleic acid molecules ashereinbefore described.

According to one aspect of this embodiment, there is providedrecombinant haemopoietin receptor encoded by a nucleic acid moleculewhich comprises a nucleotide sequence or a complementary form thereofwhich is capable of hybridising to SEQ ID NO:1 under medium stringentconditions.

In a preferred embodiment, the recombinant haemopoietin receptor isencoded by a nucleic acid molecule which comprises a nucleotide sequenceor a complementary form thereof which is capable of hybridising to SEQID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 undermedium strent conditions.

In an even more preferred embodiment, the recombinant haemopoietinreceptor is encoded by a nucleic acid molecule which comprises anucleotide sequence or complementary form thereof which hybridises undermedium stringency conditions to one or more of SEQ ID NO:1 and SEQ IDNO:4 to SEQ ID NO:11.

In still an even more preferred embodiment, the recombinant haemopoietinreceptor is encoded by a nucleic acid molecule which comprises anucleotide sequence or a complementary form thereof which hybridisesunder medium stringent conditions to each of oligonucleotides SEQ IDNO:1 and SEQ ID NO:4 to SEQ ID NO:11.

In a most preferred embodiment, the present invention is directed to arecombinant NR2 encoded by a nucleic acid molecule comprising anucleotide sequence or complementary form thereof substantially as setforth in SEQ ID NO:12 or a sequence capable of hybridising to all orpart thereof under medium stringent conditions.

According to this latter aspect of the present invention, there isprovided a recombinant NR2 having an amino acid sequence subantially asset forth in FIG. 2 [SEQ ID NO:13] or having at least about 60%similarity to all or part thereof, more preferably at least about 70%,still more preferably at least about 80% and still more preferably atleast about 90-95% or above (e.g. 96%, 97%, 98% or greater than or equalto 99%) similarly to all or part of the amino acid sequence set forth inSEQ ID NO:13.

The recombinant NR2 or a genetic sequence encoding same is preferably inisolated form meaning that a composition of matter comprises at leastabout 10%, more preferably at least about 20%, still more preferably atleast about 30-40%, even more preferably at least about 50-60%, stilleven more preferably at least about 70-80% or greater (e.g. 85%, 90or95%) of the recombinant receptor or genetic sequence encoding samerelative to other components in the composition as determined by, forexample, molecular weight, activity, nucleic acid content or compositionor other convenient means.

Reference herein to “recombinant haemopoietin receptor”, “NR2” or“leptin receptor” includes reference to derivatives thereof such asparts, fragments, portions, homologues, hybrids or analogues thereof.The derivatives may be finctional or not or may be non-functional butimmunologically interactive with antibodies to all or part of thereceptor. Derivatives of the receptor also cover agonists or antagonistsof receptor-ligand interaction. Function is conveniently defined by anability of NR2 to interact with leptin or for soluble NR2 to competewith leptin-induced activities of certain cells.

For the purposes of defining the level of stringency, reference canconveniently be made to Sambrook et al (14) which is herein incorporatedby reference where the washing steps disclosed at pages 952-957 areconsidered high stringency. A low stringency is defined herein as beingin 4-6X SSC/0.1-0.5% w/v SDS at 37-45° C. for 2-3 hours. Depending onthe source and concentration of nucleic acid involved in thehybridisation, alternative conditions of stringency may be employed suchas medium stringent conditions which are considered herein to be 1-4XSSC/0.25-0.5% w/v SDS at ≧ 45° C. for 2-3 hours or high stringentconditions considered herein to be 0.1-1X SSC/0.1% w/v SDS at ≧60° C.for 1-3 hours.

The nucleic acid molecule is preferably derivable from the human genomebut genomes and nucleotide sequences from non-human animals are alsoencompassed by the present invention. Non-human animals contemplated bythe present invention include livestock animals (e.g. sheep, cows, pigs,goats, horses, donkeys), laboratory test animals (e.g. mice, rats,guinea pigs, hamsters, rabbits), domestic companion animals (e.g. dogs,cats), birds (e.g. chickens, geese, ducks and other poultry birds, gamebirds, emus, ossriches) and captive wild or tamed animals (e.g. foxes,kangaroos, dingoes).

Preferred human genetic sequences encoding NR2 include sequences fromcells of bone marrow, brain, liver, kidney, heart, testis, stomach,lymph nodes, colon, spleen and ovary, neonatal tissue, embryonic tissue,cancer or tumour-derived tissues.

The nucleic acid molecule of the present invention may be single ordouble stranded, linear or closed circle DNA (e.g. genomic DNA), cDNA ormRNA or combinations thereof such as in the form of DNA:RNA hybrids. Thenucleic acid molecule may also include a vector such as an expressionvector component to facilitate expression of the haemopoietin recetor orits components or parts.

As stated above, the present invention further contemplates a range ofderivatives of NR2. Derivatives include fragments, parts, portions,mutants, homologues and analogues of the NR2 polypeptide andcorresponding genetic sequence. Derivatives also include single ormultiple amino acid substitutions, deletions and/or additions to NR2 orsingle or multiple nucleotide substitutions, deletions and/or additionsto the genetic sequence encoding NR2. “Additions” to amino acidsequences or nucleotide sequences include fusions with other peptides,polypeptides or proteins or fusions to nucleotide sequences. Referenceherein to “NR2” includes reference to all derivatives thereof includingfunctional derivatives or “NR2” immunologically interactive derivatives.

Analogues of NR2 contemplated herein include, but are not limited to,modification to side chains, incorporating of unnatural amino acidsand/or their derivatives during peptide, polypeptide or proteinsynthesis and the use of crosslinkers and other methods which imposeconformational constraints on the proteinaceous molecule or theiranalogues.

Examples of side chain modifications contemplated by the presentinvention include modifications of amino groups such as by reductivealkylation by reaction with an aldehyde followed by reduction withNaBH₄; amidination with methylacetimidate; acylation with aceticanhydride; carbamoylation of amino groups with cyanate;trinitrobenzylation of amino groups with 2, 4, 6trinitrobenzenesulphonic acid (TNBS); acylation of amino groups with succinic anhydrideand tetrahydrophthalic anhydride; and pyridoxylation of lysine withpyridoxal-5- phosphate followed by reduction with NaBH4.

The guanidine group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivitision, forexample, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylationwith iodoacetic acid or iodoacetamide; performic acid oxidation tocysteic acid; formation of a mixed disulphides with other thiolcompounds; reaction with maleimide, maleic anhydride or othersubstituted maleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4- chloromercuriphenylsulphonic acid,phenylmercury chloride, 2-choloromercuri-4-nitrophenol and othermercurials; carbamoylation with cyanate at akaline pH.

Tryptophan residues may be modified by, for example, oxidation withN-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residueson the other hand, may be altered by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoactic acid derivatives orN-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives duringpeptide synthesis include, but are not limited to, use of norleucine,4-amino butyric acid, 4-amino-3-hydroxy-5- phenylpentanoic acid,6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine,ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid,2-thienyl alanine and/or D- isomers of amino acids. A list of unnaturalamino acid, contemplated herein is shown in Table 1.

Crosslinkers can be used, for example, to stabilise 3D conformations,using horno-bifunctional crosslinkers such as the bifunctional imidoesters havig (CH₂)_(n) spacer groups with n=1 to n=6, glutaraldehyde,N-hydroxysuccinimide esters and hetero-bifunctional reagents whichusually contain an amino-reactive moiety such as N-hydroxysuccinimideand another group specific-reactive moiety such as maleimido or dithiomoiety (SH) or carbodiimide (COOH). In addition, peptides can beconformationally constrained by, for example, incorporation of C_(α) andN_(α)-methylamino acids, introduction of double bonds between C_(α) andC_(β) atoms of amino acids and the formation of cyclic peptides oranalogues by introducing covalent bonds such as forming an amide bondbethween the N and C termini between two side chains or between a sidechain and the N or C terminus.

These types of modifications may be important to stabilise NR2 ifadministered to an individual or for use as a diagnostic reagent.

The present invention further contemplates chemical analogues of NR2capable of acting as antagonists or agonists of NR2 or which can act asfunctional analogues of NR2. Chemical analogues may not necessarily bederived from NR2 but may share certain conformational similarities.Alternatively, chemical analogues may be specifically designed to mimiccertain physiochemical properties of NR2. Chemical analogues may bechemically synthesised or may be detected following, for example,natural product screening.

The idenification of NR2 permits the generation of a range oftherapeutic molecules capable of modulating expression of NR2 ormodulating the activity of NR2. Modulators contemplated by the presentinvention includes agonists and antagonists of NR2 expresion.Antagonists of NR2 expression include antisense molecules, ribozymes andco-suppression molecules. Agonists include molecules which increasepromoter ability or interfere with negative regulatory mechanisms.Agonists of NR2 include molecules which overcome any negative regulatorymechanism. Antagonists of NR2 include antibodies and inhibitor peptidefragmets.

TABLE 1 Non-conventional Non-conventional amino acid Code amino acidCode α-aminobutyric acid Abu L-N-methylalanine Nmalaα-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmargaminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylateL-N-methylaspartic acid Nmasp aminoisobutyric acid AibL-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmglncarboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine ChexaL-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine DargL-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine NmmetD-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine DglnL-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine NmornD-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine DileL-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysineDlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophanNmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine DpheL-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine NmetgD-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine DthrL-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-amiobutyrateMgabu D-α-methylalanine Dmala α-methylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcylcopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-a-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cylcododecylglycine Ncdod D-N-methylalanine Dnmala N-cyclooctylglycineNcoct D-N-methylarginine Dnmarg N-cyclopropylglycine NcproD-N-methylasparagine Dnmasn N-cycloundecylglycine NcundD-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-N-methylcysteine Dmncys N-(3,3-diphenylpropyl)glycine NbheD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydrodxyethyl))glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalamine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetL-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan MtrpL-α-methyltyrosine Mtyr L-α-methylvaline MvalL-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) NnbhmN-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycinecarbamylmethyl)glycine 1-carboxy-1-(2,2-diphenyl- Nmbcethylamino)cyclopropane

Other derivatives contemplated by the present invention include a rangeof glycosylation variants from a completely unglycosylated molecule to amodified glycosylated molecule. Altered glycosylation patterns mayresult from expression of recombinant molecules in different host cells.

Another embodiment of the present invention contemplates a method formodulating expression of NR2 in a human, said method comprisingcontacting the NR2 gene encoding NR2 with an effective amount of amodulator ofNR2 exepression for a time and under conditions sufficientto up-regulate or down-regulate or otherwise modulate expression ofNR2.For example, a nucleic acid molecule encoding NR2 or a derivativethereof may be introduced into a cell to enhance NR2 related activitiesof that cell. Conversely, NR2 antisense sequences (or sense sequencesfor co-suppression) such as oligonucleotides may be introduced todecrease NR2-related activies of any cell expressing the endogenous NR2gene. Ribozymes may also be used.

Another aspect of the present invention contemplates a method ofmodulating an activiy of NR2 in a human, said method comprisingadministering to said mammal a modulating effective amount of a moleculefor a time and under conditions sufficient to increase or decrease NR2activity. The molecule may be a proteinaceous molecule or a chemicalentity and may also be a derivative of NR2 or its receptor or a chemicalanalogue or truncation mutant of NR2 or its receptor.

Accordingly, the present invention contemplates a pharmaceuticalcomposition comprising NR2 or a derivative thereof or a modulator of NR2expression or NR2 activity and one or more pharmaceutically acceptablecarriers and/or diluents. These components are referred to as the activeingredients.

In this regard there is provided a pharmaceutical composition comprisinga recombinant haemopoietin recptor as hereinbefore decribed or a ligand(e.g. leptin) binding portion thereof and one or more pharmaceuticallyacceptable carrers and/or diluents.

In another embodiment, there is provided a pharmaceutical compositioncomprising a ligand (e.g. leptin) to the recombinant haemopoietinreceptor as hereinbefore described and one or more pbarmaceuticallyacceptable carriers and/or diluents.

Still a further aspect of the present invention contemplates a method oftreatment of an animal comprising administering to said animal atreatment effective amount of a recombinant haemopoietin receptor ashereinbefore described or a ligand binding portion thereof or a ligand(e.g. leptin) to said haempoietic receptor for a time and underconditions sufficient for said treatment to be substantially effected orthe conditions to be substantially ameliorated.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion or may be in the form of a cream or other formsuitable for topical application. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained for example,by the use of a coating such as licithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsuperfactants. The preventions of the action of microorganisms can bebrought about by various antbacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepaed by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredient into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

When the active ingredients are suitably protected they may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or it may be enclosed in hard or soft shell gelatincapsule, or it may be compressed into tablets, or it may be incorporateddirectly with the food of the diet. For oral therapeutic administration,the active compound may be incorporated with excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 1% by weight of active compound.The percentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 5 to about 80% of theweight of the unit. The amount of active compound in suchtherapeutically useful compositions in such that a suitable dosage willbe obtained. Preferred compositions or preparations according to thepresent invention are prepared so that an oral dosage unit form containsbetween about 0.1 ug and 2000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain thecomponents as listed hereafter: A binder such as gum, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such a sucrose, lactose or saccharin may be added or a flavouringagent such as peppermint, oil of wintergreen, or cherry flavouring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materis maybe present as coatings or to otherwise modify the physical form of thedosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavouring such as cherry or orange flavour. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound(s) may be incorporated intosustained-release preparations and formulations.

The present invention also extends to forms suitable for topicalapplication such as creams, lotions and gels.

Pharmaceutically acceptable carriers and/or diluents include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, use thereof in the therapeuticcompositions is contemplated. Supplementary active ingrdients can alsobe incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with thereqired pharmaceutical carrier. The specification for the novel dosageunit forms of the invention are dictated by and directly dependent on(a) the unique characteristics of the active material and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active material for the treatment ofdisease in living subjects having a diseased condition in which bodilyhealth is impaired.

The principal active ingredient is compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form as hereinbeforedisclosed. A unit dosage form can, for example, contain the principalactive compound in amounts ranging from 0.5 μg to about 2000 mg.Expressed in proportions, the active compound is generally present infrom about 0.5 μg to about 2000 mg/ml of carrier. In the case ofcompositions containing supplementary active ingredients, the dosagesare determined by reference to the usual dose and manner ofadministration of the said ingredients.

The pharmaceutical composition may also comprise genetic molecules suchas a vector capable of transfecting target cells where the vectorcarries a nucleic acid molecule capable of modulating NR2 expression orNR2 activity. The vector may, for example, be a viral vector.

Still another aspect of the present invention is directed to antibodiesto NR2 and its derivatives or its ligands (e.g. leptin). Such antibodiesmay be monoclonal or polyclonal and may be selected from naturllyoccurring antibodies to NR2 or may be specifically raised to NR2 orderivatives thereof. In the case of the latter, NR2 or its derivativesmay first need to be associated with a carrier molecule. The antibodiesand/or recombinant NR2 or its derivatives of the present invention areparticularly useful as therapeutic or diagnostic agents.

For example, NR2 and its derivatives can be used to screen for naturallyoccurring antibodies to NR2. These may occur, for example in someautoimmune diseases. Alternatively, specific antibodies can be used toscreen for NR2. Techniques for such assays are well known in the art andinclude, for examnple, sandwich assays and ELISA. Knowledge ofNR2 levelsmay be important for diagnosis of certain cancers or a predisposition tocancers or for monitoring certain therapeutic protocols.

Antibodies to NR2 of the present invention may be monoclonal orpolyclonal. Alternatively, fragments of antibodies may be used such asFab fragments. Furethermore, the present invention extends torecombinant and synthetic antibodies and to antibody hybrids. A“synthetic antibody” is considered herein to include fragments andhybrids of antibodies. The antibodies of this aspect of the presentinvention are particularly useful for immunotherapy and may also be usedas a diagnostic tool for assessing the receptor or receptor-ligandinteraction or monitoring the program of a therapeutic regimin.

For example, specific antibodies can be used to screen for NR2 proteins.The latter would be important, for example, as a means for screening forlevels of NR2 in a cell extract or other biological fluid or purifyingNR2 made by recombinant means from culture supernatant fluid. Techniquesfor the assays contemplated herein are known in the art and include, forexample, sandwich assays and ELISA.

It is within the scope of this invention to include any secondantibodies (monoclonal, polyclonal or fragments of antibodies orsynthetic antibodies) directed to the first mentioned antibodiesdiscussed above. Both the first and second antibodies may be used indetection assays or a first antibody may be used with a commerciallyavailable anti-immunoglobulin antibody. An antibody as contemplatedherein includes any antibody specific to any region of NR2.

Both polyclonal and monoclonal antibodies are obtainable by immunizationwith the enzyme or protein and either type is utilizable forimmunoassays. The methods of obtaining both types of sera are well knownin the art. Polyclonal sera are less preferred but are relatively easilyprepared by injection of a suitable laboratory animal with an effectiveamount of NR2, or antigenic parts thereof, collecting serum from theanimal, and isolating specific sera by any of the known immunoadsorbenttechniques. Although antibodies produced by this method are utilizablein virtally any type of immunoassay, they are generally less favouredbecause of the potential heterogeneity of the product.

The use of monoclonal antibodies in an immunoassay is particularlypreferred because of the ability to produce them in large quantities andthe homogeneity of the product. The preparation of hybridoma cell linesfor monoclonal antibody production derived by fusing an immortal cellline and lymphocytes sensitized against the immunogenic preparation canbe done by techniques which are well known to those who are skilled inthe art.

Another aspect of the present invention contemplates a method fordetecting NR2 in a biological sample from a subject said methodcomprising contacting said biological sample with an antibody specificfor NR2 or its derivatives or homologues for a time and under conditionssufficient for an antibody-NR2 complex to form, and then detecting saidcomplex.

The presence of NR2 may be accomplished in a number of ways such as byWestern blotting and ELISA procedures. A wide range of immunoassaytechniques are available as can be seen by reference to U.S. Pat. Nos.4,016,043, 4,424,279 and 4,018,653. These, of course, includes bothsingle-site and two-site or “sandwich” assays of the non-competitivetypes, as well as in the traditional competitive binding assays. Theseassays also include direct binding of a labelled antibody to a target.

Sandwich assays are among the most useful and commonly used assays andare favoured for use in the present invention. A number of variations ofthe sandwhich assay technique exist, and all are intended to beencompassed by the present invention. Briefly, in a typical forwardassay, an unlabelled antibody is immobilized on a solid substrate andthe sample to be tested brought into contact with the bound molecule.After a suitable period of incubation, for a period of time sufficientto allow formation of an antibody-antigen complex, a second antibodyspecific to the antigen, labelled with a reporter molecule capable ofproducing a detectable signal is then added and incubated, allowing timesufficient for the formation of another complex ofantibody-antigen-labelled antibody. Any unreacted material is washedaway, and the presence of the antigen is determined by observation of asignal produced by the reporter molecule. The results may either bequalitative, by simple observation of the visible signal, or may bequantitated by comparing with a control sample containing known amountsof hapten. Variations on the forward assay include a simultaneous assay,in which both sample and labelled antibody are added simultaneously tothe bound antibody. These techniques are well known to those skilled inthe art, including any minor variations as will be readily apparent. Inaccordance with the present invention the sample is one which mightcontain NR2 including cell extract, tissue biopsy or possibly serum,saliva, mucosal secretions, lymph, tissue fluid and respiratory fluid.The sample is, therefore, generally a biological sample comprising abiological fluid, cell extract, bone marrow or lymph, tissue extract(e.g. from kidney, liver, spleen, etc), fermentation fluid andsupernatant fluid such as from a cell culture and cell conditionedmedium.

In the typical forward sandwich assay, a first antibody havingspecificity for the NR2 or antigenic parts thereof, is either covalentlyor passively bound to a solid surface. The solid surface is typicallyglass or a polymer, the most commonly used polymers being cellulose,polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.The solid supports may be in the form of tubes, beads, discs ofmicroplates, or any other surface suitable for conducting animmunoassay. The binding processes are well-known in the art andgenerally consist of cross-linking covalently binding or physicallyadsorbing, the polymer-antibody complex is washed in preparation for thetest sample. An aliquot of the sample to be tested is then added to thesolid phase complex and incubated for a period of time sufficient (e.g.2-40 minutes) and under suitable conditions (e.g. 25° C.) to allowbinding of any subunit present in the antibody. Following the incubationperiod, the antibody subunit solid phase is washed and dried andincubated with a second antibody specific for a portion of the hapten.The second antibody is linked to a reporter molecule which is used toindicate the binding of the second antibody to the hapten.

An alternative method involves immobilizhng the target molecules in thebiological sample and then exposing the immobilized target to specificantibody which may or may not be labelled with a reporter molecule.Depending on the amount of target and the strength of the reportermolecule signal, a bound target may be detectable by direct labellingwith the antibody. Alternatively, a second labelled antibody, specificto the first antibody is exposed to the target-first antibody complex toform a target-first antibody-second antibody tertiary complex. Thecomplex is detected by the signal emitted by the reporter molecule.

By “reporter molecule” as used in the present specification, is meant amolecule which, by its chemical nature, provides an analyticallyidentifiable signal which allows the detection of antigen-boundantibody. Detection may be either qualitative or quantitative. The mostcommonly used reporter molecules in this type of assay are eitherenzymes, fluorophores or radionuclide containing molecules (i.e.radioisotopes) and chemiluminescent molecules. In the case of an enzymeimmunoassay, an enzyme is conjugated to the second antibody, generallyby means of glutaraldehyde or periodate. As will be readily recognized,however, a wide variety of different conjugation techniques exist, whichare readily available to the skilled artisan. Commonly used enzymesinclude horseradish peroxidase, glucose oxidase, beta-galactosidase andalkaline phosphatase, amongst others. The substrates to be used with thespecific enzymes are generally chosen for the production, uponhydrolysis by the corresponding enzyme, of a detectable colour change.Examples of suitable enzymes include alkaline phosphatase andperoxidase. It is also possible to employ fluorogenic substrates, whichyield a fluorescent product rather than the chromogenic substrates notedabove. In all cases, the enzyme-labelled antibody is added to the firstantibody hapten complex, allowed to bind, and then the excess reagent iswashed away. A solution containing the appropriate substrte is thenadded to the complex of antibody-antigen-antibody. The substrate willreact with the enzyme linked to the second antibody, giving aqualitative visual signal, which may be furter quantitated, usuallyspectrophotometrically, to give an indication of the amount of haptenwhich was present in the sample. “Reporter molecule” also extends to useof cell agglutination or inhibition of agglutination such as red bloodcells on latex beads, and the like.

Alternatively, fluorescent compounds, such as fluorescein and rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labelled antibody adsorbs the light energy,inducing a state to excitability in the molecule, followed by emissionof the light at a characteristic colour visually detectable with a lightmicroscope. As in the EIA, the fluorescent labelled antibody is allowedto bind to the first antibody-hapten complex. After washing off theunbound reagent, the remaining tertiary complex is then exposed to thelight of the appropriate wavelength the fluorescence observed indicatesthe presence of the hapten of interest Immunofluorescene and EIAtechniques are both very well established in the art and areparticularly preferred for the present method. However, other reportermolecules, such as radioisotope, chemiluminescent or bioluminescentmolecules, may also be employed.

The present invention also contemplates genetic assays such as involvingPCR analysis to detect NR2 gene or its derivatives. Alternative methodsor methods used in conjunction include direct nucleotide sequencing ormutation scanning such as single stranded conformation polymorphomsanalysis (SSCP) as specific oligonucleotide hybridisation, as methodssuch as direct protein truncation tests. Such genetic tests may beimportant, for example, in genetic screening of animals (e.g. humans)for non-expression or substantial absence of expression or expression ofmutant forms of NR2 leading to conditions such as obesity or othereffects of leptin-receptor interaction.

The nucleic acid molecules of the present invention may be DNA or RNA.When the nucleic acid molecule is in DNA form, it may be genomic DNA orcDNA. RNA forms of the nucleic acid molecules of the present inventionare generally mRNA.

Although the nucleic acid molecules of the present invention aregenerally in isolated form, they may be integrated into or ligated to orotherwise fused or associated with other genetic molecules such asvector molecules and in particular expression vector molecules. Vectorsand expression vectors are generally capable of replication and, ifapplicable, expression in one or both of a proklyotic cell or aeukaryotic cell. Preferably, prokaryotic cells include E. Col. Bacillussp and Pseudomonas sp. Preferred eukaryotic cells include yeast, fungal,mammalian and insect cells.

Accordingly, another aspect of the present invention contemplates agenetic construct comprising a vector portion and a mammalian and moreparticularly a human NR2 gene portion, which NR2 gene portion is capableof encoding an NR2 polypeptide or a functional or immunologicallyinteractive derivative thereof.

Preferably, the NR2 gene portion of the genetic construct is operablylinked to a promoter on the vector such that said promoter is capable ofdirecting expression of said NR2 gene portion in an appropriate cell.

In addition, the NR2 gene portion of the genetic consruct may compriseall or part of the gene fused to another genetic sequence such as anucleotide sequence encoding glutathione-S- transferase or part thereofor a cytokine or another haempoietic receptor. Hybrid receptor moleculesare particularly useful in the development of multi functionaltherapeutic and diagnostic agents.

The present invention extends to such genetic constructs and toprokaryotic or eukaryotic cells comprising same.

The present invention also extends to any or all derivatives of NR2including mutants, part, fragments, portions, homologues and analoguesor their encoding genetic sequence including single or multiplenucleotide or amino acid substitutions, additions and/or deletions tothe naturally occurring nucleotide or amino acid sequence.

The NR2 and its genetic sequence of the present invention will be usefulin the generation of a range of therapeutic and diagnostic reagents andwill be especially useful in the detection of a correponding ligand. Forexample, recombinant NR2 may be bound or fused to a reporter moleculecapable of producing an identifiable signal, contacted with a biologicalsample putatively containing a ligand and screening for binding of thelabelled NR2 to the ligand. Alternatively, labelled NR2 may be used toscreen expression libraries of putative ligand genes or functional partsthereof.

In another embodiment, the NR2 is first immobilised. According to thisembodiment, there is provided a method comprising contacting abiological sample containing a putative ligand with said haempoieticreceptor or a ligand binding portion thereof immobilised to a solidsupport for a time and under conditions sufficient for a complex to formbetween said receptor and said ligand if said ligand is present in saidbiological sample, eluting bound ligand and isolating same.

Soluble NR2 polypeptides are also contemplated to be useful in thetreatment of disease, injury or abnormality in the nervous system, e.g.in relation to central or peripheral nervous system to treat CerebralPalsy, trauma induced paralysis, vascular ischaemia associated withstroke, neuronal tumours, motoneurone disease, Parkinson's disease,Huntington's disease, Alzheimer's disease, Multiple Sclerosis,peripheral neuropathies associated with diabetes, heavy metal or alcoholtoxicity, renal failure and infectious diseases such as herpes, rubella,measles, chicken pox, HIV or HTLV-1. The NR2 polypeptides may also beimportant for regulating cytokine activity such as leptin activity,modulating haempoiesis and/or regulating or modulating adipose tissue.

As stated above, the NR2 or its ligand of the present invention or theirfunctional derivatives may be provided in a pharmacuetical compositiontogether with one or more pharmaceutically acceptable carriers and/ordiluents. In addition, the present invention contemplates a method oftreatment comprising the administration of an effective amount of NR2 ofthe present invention. The present invention also extends to antagonistsand agonists of NR2 and/or its ligand and their use in therapeuticcompositions and methodologies.

A further aspect of the present invention contemplates the use of NR2 orits functional derivatives in the manufacture of a medicament for thetreatment ofNR2 mediated conditions defective or deficient.

The present invention is further described with reference to thefollowing non-limiting Examples.

The following single and three letter abbreviations for amino acidresidues are used in the specification:

Three-letter One-letter Amino Acid Abbreviation Symbol Alanine Ala AArginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V Any residue Xaa X

TABLE 2 SEQUENCE OF OLIGONUCLEOTIDES OLIGONUCLEOTIDE SEQUENCE SEQ ID NOsHYB2 5′(A/G)CTCCA(A/G)TC(A/G)CTCCA-3′ 1 T3 5′-TAATACGACTCACTATAGGGAGA-3′2 T7 5′-ATTAACCCTCACTAAAGGGA-3′ 3  721 5′-ACTAGCAGGGATGTAGCTGAG-3′ 4 722 5′-CTCAGCTACATCCCTGCTAGT-3′ 5  761 5′-CTGCTCCTATGATACCT-3′ 6  8755′-CCTCTTCCATCTTATTGCTTGG-3′ 7  939 5′-ATCGGTCGTGACATACAAGG-3′ 8 10565′AGCTAAGCTTTCTAGATATCCAATTACTCCTTGGAGA-3′ 9 10925′-AGCTTCTAGATCAATCACTCTGGTGTTTTTCAAT-3′ 10 10945′-AGCTTCTAGATCAAACTTTTATATCCATGACAAC-3′ 11

EXAMPLE 1 CLONING OF A HUMAN NR2 (HAEMOPOIETIN RECEPTOR) cDNA

A cDNA library constructed from mRNA from a the bone marrow of a patientrecovering from chemotherapy was constructed by C.G. Begley, CancerResearch Unit, WEHI in 1ZAP (Stratagene, CA, USA) were used to infectEscherichia coli of the strain LE392. Infected bacteria were grown ontwenty 150 mm agar plates, to give approximately 50,000 plaques perplate. Plaques were then transferred to duplicate 150 mm diameter nylonmembranes (Colony/Plaque Screen™, NEN Research Products, MA, USA),bacteria were lysed and the DNA was denatured fixed by autoclaving at100° C. for 1 min with dry exhaust. The filters were rinsed twice in0.1%(w/v) sodium dodecyl sulfate (SDS), 0.1×SSC (SSC is 150 mM sodiumchloride, 15 mM sodium citrate dihydrate) at room temperature andpre-hybridised overnight at 42° C. in 6×SSC containing 2 mg/ml bovineserum albumin, 2 mg/ml Ficoll, 2 mg/ml polyvinylpyrrolidone, 100 mM ATP,10 mg/ml tRNA, 2 mM sodium pyrophosphate, 2 mg/ml salmon sperm DNA, 0.1%SDS and 200 mg/ml sodium azide. The prehybridisation buffer was removed.1.2 mg of the degenerate oligonucleotides for hybridisation (HYB2; Table2 above) were phosphorylated with T4 polynucleotide kinase using 960 mCiof g³²P-ATP (Bresatec, S.A., Australia). Unincorporated STP wasseparated from the labelled oligonucleotide using a pre-packed gelfiltration column (NAP-5; Pharmacia, Uppsala, Sweden). Filters werehybridised overnight at 37° C. in 80 ml of the prehybridisation buffercontaining and 10⁶-10⁷ cpm/ml of labelled oligonucleotide. Filters werebriefly rinsed twice at room temperature in 6×SSC, 0.1%(v/v) SDS, twicefor 30 min at 45° C. in a shaking waterbath containing 1.51 of the samebuffer and then briefly in 6×SSC at room temperature. Filters were thenblotted dry and exposed to autoradiographic film −70° C. usingintensifying screens, for 7-14 days prior to development.

Plaques that appeared to hybridise to the probe on duplicate filterswere picked and eluted for 2 days at 4° C in 0.5 ml of 100 mM NaCl, 10mM MgCl₂, 10 mM Tris.HCl pH7.4 containing 0.5%(w/v) gelatin and 0.5%(v/v) chloroform. 5 ml aliquots of each eluate was used as the substratefor two PCR reactions containing 5 ml of 10× concentrated PCR buffer(Boehringer Mannheim GmbH, Mannheim, Germany), 1 ml of 10 mM dATP, dCTP,dGTP and dTTP, 2.5 ml of the oligonucleotides HYB2 and either T3 or T7at a concentration of 100 mg/ml, 0.5 ml of Taq polymerase (BoehringerMannheim GmbH) and water to a final volume of 50 ml. PCR was carried outin a Perkin-Elmer 9600 by heating the reactions to 96° C. for 2 min andthen for 25 cycles at 96° C. for 30 sec, 55° C. for 30 sec and 72° C.for 2 min. The reactions were then electrophoresed on a 1 %(w/v) lowmelting point agarose gel in TAE. Any positive products were excised,the gel slice was melted and 2 ml was used as the substrate for a secondPCR reaction using conditions identical to the first. The product fromthe second reaction was purified using an ultrafree-MC centrifugalfiltration unit (Millipre Corp.) by centrifugation for 15 min at 2000 gin an eppendorf centrifuge, adding 0.5 ml of 10 mM Tris.HCl, 1 mM EDTApH8 and recentrifuging. This procedure was repeated three times and theDNA was recovered in 50 ml of 10 mM Tris.HCl, 1 mM EDTA pH8.

Approximaely 500 ng of DNA from each PCR reaction was sequenced using afmol sequencing kit (Promega Corporation, WI, USA), according to themanufacturer's instructions with the ³³P-labelled oligonucleotide primerHYB2. The products were resolved on a 6% w/v polyacrylamide gel and thesequence of each clone was analysed using the Blast database comparisonprograms and the translation function of the Wisconsin suite of DNAprograms. The sequence of the PCR product derived from the primaryplaque eluate number CF.32 appeared to be novel since it had nohomologues in the databases of DNA sequences that were searched, andupon inspection of the sequence of the conceptually translated productsappeared also to be a member of the haemopoietin rector family. Thisclone was given the name of new rcceptor 2 or NR2.

The positively hybridising bacteriophage present in the eluate from theprimary NR2-CF-32-1 was purified using a second round of screeningperformed in a manner identical to the first, except that plaques weregrown on smaller, 82 mm, plates of agar. Once purified DNA, the positivecDNA cloned into the plasmid pBluescript was excised from the λ-ZAP IIbacteriophage according to the manufacturer's instructions (Statagene).A CsCl purified preparation of the DNA was made and this was sequencedon both strands. Sequencing was performed using an Applied Biosystemsautomated DNA sequencer, with fluorescent dideoxynucleotide analoguesaccording to the manufacturer's instructions. The DNA sequence wasanalysed using software supplied by Applied Biosystems.

EXAMPLE 2 ISOLATION OF ADDITIONAL NR2 cDNAS

NR2-CF.32 did not appear to contain the entire coding region of thenovel receptor. In order to identify other cDNA libraries containingcDNA clones for NR2 we performed PCR upon 1 ml aliquots ofλ-bacteriophage cDNA libraries made from mRNA from various human tissuesand using oligonucleotides 722 and 761, designed from NR2-CF-32-1, asprimers. The oligonucleotides are defined in Table 2, above. Reactionscontained the same elements as described above and were performed in anidentical manner. In addition to the orginal library, five other cDNAlibraries appeared to contain NR2 cDNAs. These were screened using a³²P-labelled oligonucleotide 721 and 761 designed from the 5′-end andthe 3′ end of the sequence derived from NR2-CF.32, using conditionsidentical to those described in section (i) except that filters werewashed at 55° C. rather than 45° C. Again, as described in section (i),positively hybridising plaques were purified, the cDNAs were recoveredand cloned into plasmids pBluescript II or pUCl19. Ten independent cDNAclones were sequenced on both stands. Further clones were isolated in asimilar manner by screening libraries with oligonucleotide 875 and 939.

The extent of each clone is illustrated in FIG. 1 and a compositesequnce is shown in FIGS. 2A to 2K. NR2 clearly has all the features ofa member of the haemopoietin rcceptor family.

EXAMPLE 3 ANALYSIS OF TIRE EXPRESSION PATTERN OF NR2 mRNA

Northern blots of mRNA from various human tissues and cell lines werehybridised with a random-primed human NR2 cDNA fragment from theinternal EcoR I site to the Hpa I site (FIG. 1). Using the protocoldescribed previously by Hilton et al. (15), two human NR2 mRNA specieswere observed to be expressed at a low level in a range of adulttissues, and at higher levels in foetal tissues such as the lung andliver. FIG. 9 shows expression ofNR2 in various mouse tissues usinghuman NR2 cDNA as probe. Interestingly among a series of humanhaemopoietin cell lines the megakaryotic cell line MEGO1 expressed highlevels of NR2 mRNA suggesting that NR2 and its cognate ligand may play arole in the regulation of the megakaryocyte proliferation,differentation and/or function.

EXAMPLE 4 GENERATION OF PLASMIDS DIRECTING THE EXPRESSION OF FULL-LENGTHAND SECRETED FORMS HUMAN NR2

Since antibodies to NR2 were not available to monitor expression,constructs were engineered to express full length and two solubleversions of NR2 with an N-terminal “FLAG” epitope (InternationalBiotechnologies/Eastman Kodak, New Haven CT). First, a derivative of themammalian expression vector pEF-BOS was generated so that it containedDNA encoding the signal sequence of murine IL-3(MVLASSTTSIHTMLLLLLMLFHLGLQASIS [SEQ ID NO. 14]) and the FLAG epitope(DYKDDDDK [SEQ ID NO 15]) followed by a unique Xba I cloning site. Thisvector was named pEF/IL3SIG/FLAG.

The 5′ end of the mature NR2 coding region was generated by PCR usingprimers 1056 and 721 on clone 60-58-7 (FIG. 1). The EcoR I/Hpa Ifragment of clone 60-55-7-6 containing the 3′ end of the NR2 codingregion and a portion of the 3-untranslated region was cloned into theEcoR I/SmaI digested pBluescript (FIG. 1). This construct was digestedwith Hind III and EcoR I and into it was cloned the 5′-NR2 PCR productdigested with the same enzymes. The resulting construct was digestedwith Xba I to yield a fragment which contained the coding region ofhuman NR2 from Y26 to the natural last amino acid L897 (FIG. 1) and asegment of 3′-untranslated region and was cloned into the Xba I site ofpEF/IL3SIG/FLAG to give pEF/IL3SIG/FLAG/NR2/897. A soluble derativeofhuman NR2 was also engineered. PCR was carried out either usingprimers 1056 and 1092 to amplify the predicted mature coding region ofthe extracellular portion of human NR2 (Y26 to D839; FIG. 1). The PCRproducts were digested with Xba I and subcloned into Xba I digestedpEF/EL3SIG/FLAG to give pEF/IL3SIG/FLAG/NR2/839. The identity of eachconstruct was confirmed by dideoxy sequencing.

EXAMPLE 5 TRANSIENT EXPRESSION OF FULL LENGTH AND SECRETED FORMS OFHUMAN NR2 IN COS CELLS

In order to confirm that full length and soluble NR2 could be producedusing the expression vectors pEF/IL3SIG/FLAG/NR2/897 andpEF/IL3SIG/FLAG/NR2/839, COS cells were transiently transfected withthese constructs. Briefly, COS cells from a confluent 175 cm2 tissueculture flask were resuspended in PBS and electroporated (BioRad Genepulser; 500 mF, 300 V) with 20 mg of uncut pEF/IL3SIG/FLAG/NR2/897 orpEF/IL3SIG/FLAG/NR2/839 in a 0.4 cm cuvette (BioRad). After 2 to 3 daysat 37° C. in a fully humidified incubator containing 10% v/v CO₂ in aircells were used for analyses of protein expression. Conditioned mediumwas collected by centrifugation and stored sterile at 4° C. Cells werealso harvested and lysed for 5 min in 500 ml of 50 mM Tris.HCl pH7.4containing 150 mM NaCl, 2 mM EDTA and 1% w/v sodium deoxycholate and0.2% w/v SDS. 15 ml of anti-FLAG M2 affinity gel (InternationalBiotechnologies/Eastman Kodak, New Haven CT) was then added to the cellextract or to 1 ml of conditioned medium and precipitation was carriedout overnight at 4° C. The affinity gel was then washed three times incold PBS and the precipitated protein was eluted by resuspending the gelin 80 ml of 100 mM sodium phosphate pH7.2, 10 mM EDTA, 0.1% w/v SDS and1% 2- mercaptoethanol and boiling for 5 min. The supernatant was removedand 8 ml of 10% b-octyl glucoside was added. One half of each sample wasincubated for 16 hours with 0.6 U of N-Glycanase-F(Boehringer-Mannheim), while the remainder was left untreated. An equalvolume of 2x SDS-PAGE sample buffer was added to the samples which werethen boiled and electrophoresed on pre-cast 4-15% w/v polyacrylamidegels (BioRad). The resolved proteins were then electroblotted ontoImmobolon membranes, which were then blocked with 5% w/v skim milk, 0.1%v/v Tween 20 in PBS, rinsed and incubated with 5 ml of anti-FLAG M2antibody in 2.5 ml of PBS containing 0.1% v/v Tween 20, rised andincubated with peroxidase-conjugated human anti-mouse Ig in 5% w/v skimmilk, 0.1% v/v Tween 20 in PBS, rinsed and incubated with ECL reagentfor 1 min. Filters were then blotted dry and exposed to autoradiogrgphicfilm for 1 mn.

COS cells that were mock tansfected contained no reactive protein, whileCOS cells transfected with pEF/IL3SIG/FLAG/NR2/897 expressed animmunoreactive protein of between 120,000 and 140,000 molecular weight.Deglycosylation with N-Glycanase-F resulted in a reduction in theapparent molecular weight to approximately 110,000 close to thatpredicted from the cDNA sequence ofNR2. The immunoreactivity observedwas completely inhibited by inclusion of an excess of the FLAG peptideduring the immunoprecipitation step. No specific immunoreactive proteinscould be detected in the medium conditioned by COS cells transfectedwith pEF/IL3SIG/FLAG/NR2/897. In contrast immunoreactive proteins werefound in the medium and the cell pellet of COS cells transfected withDNA encoding the secreted form of NR2-pEF/IL3SIG/FLAG/NR2/839. Thesecreted form of NR2, as predicted, exhibited a lower apparent molecularweight than full length NR2, 110,000 to 120,000. This again decreasedupon deglycosylation, to approximately 100,000.

COS cells transfected with pEF/IL3SIG/FLAG/NR2/897 were also examinedfor cell surface expression of NR2 by immunofluoresence staining. 5×10⁵COS cells were resuspended in 100 ml of PBS containing 5% fetal calfserum and incubated with FITC-conjugated anti-FLAG M2 antibody for 45min on ice, the cells were fixed and examined using a fluoresencemicroscope. No positive cells were observed in mock transfected samples,while approximately 10% of COS cells transfected withpEF/IL3SIG/FLAG/NR2/897 stained brightly positive. This data wasconsistent with the expected transient transfection efficiency of COScells using electroporation.

EXAMPLE 6 STABLE EXPRESSION OF FULL LENGTH HUMAN NR2

As described below certain routes to the identification of the NR2ligand require stable expression of full-length NR2 in haemopoietin celllines and the production and purification of large (mg) amounts ofsecreted NR2. Stable transfection of the pEF/IL3SIG/FLAG/NR2/897 andpEF/IL3SIG/FLAG/NR2/839 plasmids was achieved by electroporation.Briefly, the plasmids were linearised by digestion with the restrictionenzyme Aat II. 20 mg of the linearised pEF/IL3SIG/FIAG/NR2/897 plasmidand 2 mg of pPGKpuropA, pPGKneopA or pPGKhygropA (plasmids directing theexpression of the puromycin, neomycin and hygromycin resistance genes)were electropoated into 4×10⁶ parental Ba/F3 cells, Ba/F3 cellsengineered to express human gpl130 with or without coexpression of thehuman LIF receptor, Ba/F3 cells expressing the human b-chain common tothe IL-3, IL-5 and GM-CSF receptors, Ba/F3 cells expressing the humanIL-2 receptor b- and g-chains, CTLL cells or CHO cells. Briefly, cellswere washed twice in ice-cold PBS and resuspended in PBS at 5×10⁶ perml. 4×10⁶ cells were aliquoted into 0.4 mm electoporation cuvettes withthe DNA. DNA and cells were incubated for 10 min on ice andelectroporated at 270 V and 960 mF in a Bio-Rad Gene-Pulser (Bio-RadLaboratories, CA, USA). The cells were mixed with 1 ml of culturemedium, centrifuged through 3 ml of FCS and resuspended in 100 ml ofculture medium. Cells were then aliquoted into four 24 well . After twodays, selection was commenced by the addition puromycin to a concentionof 20 mg/ml, G418 to a concentration of 11.2 mg/ml or hygromycin to aconcentration of 1 mg/ml. After 10-14 days, clones of proliferting cellswere transferred to flasks and after expansion were tested for receptorexpression.

FACS analysis using the anti-FLAG M2 antibody (FIG. 3) illustrates thatBa/F3 cells transfected with the pEF/IL3SIG/FLAG/NR2/897 express NR2 onthe cell surface. Similar results have been obtained with other celllines. As with COS cells, CHO cells transfected withpEF/IL3SIG/FLAG/NR839 secrete the NR2 extracellular domain. Theextracellular domain of NR2 has been purified on an anti-FLAG M2antibody affinity column using the FLAG peptide as the means of elution.This results in a high degree of purification of the NR2 extacellulardomain as seen in the silver-stained poly-acrylamide gel illustrated inFIG. 4.

EXAMPLE 7 STRATEGIES FOR ISOLATION OF THE LIGAND FOR NR2

The stable expression of full-length and secreted NR2 enables steps tobe taken to generate specific monoclonal antibodies to NR2 and allows anumber of strategies to be employed to identify the cognate ligands ofNR2.

(a) Expression of NR2 in factor dependent cell lines;

A variety of haemopoietin cell lines have been described which aredependent on the presence of exogenous growth factor for survival andproliferation in vitro. Among these are the murine cell lines Ba/F3,FDCP-1, 32D, CTLL, NFS-60, B6SutA, DA-1 and DA-3 and the human celllines M07 and TF-1. FLAG-tagged murne and human NR2 may be stablyexpressed in each of these cell lines. The capacity of mediumconditioned by a variety of murine and human cell lines and tissues tostimulate the survival and division of factor dependent cell linesexpressing NR2 will be compared to the ability of the same medium tostimulate parental cell lines that do not express NR2. Medium that showsa greater ability to stimulate the proliferation cells expressing NR2will be considered as a potential source of NR2.

NR2 has also been co-expressed in Ba/F3 cells with the LIF receptora-chain and gpl130, with the IL-2 receptor b- and g-chains of the IL-2receptor and with the common b-chain ofthe IL-3, IL-5 and GM-CSFreceptors. Again conditioned medium will be tested for their ability tostimulate the proliferation of these cell lines.

(b) Identification of the NR2 ligand using the Cytosensor;

The haemopoietin cell lines expressing NR2 described above andadditional non-haemopoietin cell lines engineered to express NR2 will beused in conjunction with the Cytosensor screen conditioned medium forthe presence of a ligand capable of altering cellular ion fluxes.Positive conditioned medium will be considered as a potential source ofNR2 ligands.

(c) Selection of Ba/F3 cells expressing the NR2 ligand;

Ba/F3 cells expressing NR2 with or without additional receptorcomponents will be mutated with EMS or with a retrovirus. Mutants thatare capable of proliferation in the absence of added growth factor willbe selected. The medium from such clones will then be tested for theirability to stimulate the proliferation of Ba/F3 cells expresing NR2 withor without additional receptor components compared with thecorresponding Ba/F3 cells that do not express NR2. Positive conditionedmedium will be considered as a potential source of the NR2 ligand.

(d) Exprssion of NR2 in cell lines that may be induced to differentiate;

Similar experiments may be performed by expressing FLAF-tagged NR2 incells that may be induced to differentiate by cytokines. Such cellsinclude the murine lines M1 and WEHI- 3BD+ and the human lines HL-60 andU937. The capacity of medium conditioned by a variety of murine andhuman cell lines and tissues to induce the differentiation of such celllines expressing NR2 will be compared to the ability of the same mediumto stimulate parental cell lines that do not express NR2. Medium thatshows a greater ability to stimulate the differentiation of cellsexpressing NR2 will be considered as a potential source ofNR2 ligand.

(e) Use of secreted NR2 extracelluar domain as a probe on the Biosensor,

Purified excellular domain of NR2 has been obtained and is beingimmobilized on the surface of a Biosensor chip. Medium conditioned by avariety of murine and human cell lines and tissues will be passed acrossthe chip and specific changes in the surface plasmon resonance will benoted. Positive medium will be considered as a potential source of NR2ligand.

(f) Use of secreted NR2 extracellular domain as the basis of an affinitycolumn;

Purified extracellular domain of NR2 has been obtained and is beingimmobilized using a variety of chemistries. Affinity columns will beconstructed and medium conditioned by a variety of murine and human celllines and tissues will be passed through. Proteins that bind to thecolumn will be considered to be candidate NR2 ligands and will befurther characterised.

EXAMPLE 8 HUMAN LEPTIN

A human leptin cDNA (16)was cloned into the peFBOS expression vector(17) in frame with the interleukin-3 leader sequence followed by theFLAG™ epitope sequence (18). CHO cells were tansfected with this vectorby electroporation and supernatant harvested from exponentially growingcultures. The supernatant was concentrated over a YM-10 membrane(10-fold) and then applied to an affinity column containing immobilisedanti-FLAG™ antibody M2. The column was eluted with FLAG™ peptideaccording to the manufacturer instructions (Eastman Koda, Rochester,NY). The monomeric form of human leptin was purifed by gel filtrationchromatography on a Superose 12 column (Pharmacia, Uppsala, Sweden) andexchanged into 20 mM phosphate buffered (pH7.4) saline (0.15 M)containing 0.02% v/v Tween 20 and 0.02% w/v sodium azide (PBS) by gelfiltration on Sephadex G-25 M (PD-10) columns (Pharmacia). Human leptinwas iodinated with ¹²⁵I using a modified iodine monochloride method (19)to a specific radioactivity of approximately 10⁷ cpm/pmole and exchangedinto PBS as above.

EXAMPLE 9 BINDING OF ¹²⁵I HUMAN LEPTIN TO CELLS EXPRESSING NR2 OR TOSOLUBLE NR2

Cos-hNR2 are COS-7 cells electroporated with peFBOX-hNR12 and harvestedat 3 days (5×10⁴ cells used per point).

Ba/F3-hNR2 are Ba/F3 cells stably transfected with peFBOS-hNR2 (9×10⁵cells used per point).

Solh NR2 is a soluble form of human NR2 purified by anti-FLAG™ affinitychromatography from the supernatant (48 hr) of COS cells tranfectd withpeFBOS-solh NR2 (approx. 0.1 μg/ml final concentration in bindingassay).

For cells, the total reaction volume was 100 μl in RPMI-mediumcontaining 10 mM Hepes pH7.4 and 10% v/v foetal calf serum (RHF). Thereaction mixture also contained ¹²⁵I h leptin 0-6×10⁵ cpm as indicatedwith or without unlabelled h leptin (approx. 1 μg/ml).

The mixture was incubated for 1-1.5 hr at 23° C. and then layered over200 μl cold foetal calf serum in small, tapered centrifuge tubes (Elkay,Melbourne) and centrifuged at 12000 g for 10 sec. The cell pellet wasremoved by cutting tubes with a scalpel blade and the cell bound(pellet) radioactivity and the unbound radioactivity (the rest of thetube) were separately counted in a Packard Υ-counter. Specifically bound¹²⁵I h leptin was determined as the difference in counts betweenotherwise identical tubes that contained or did not contain theunlabelled excess h leptin. The data were plotted as saturation curves(specifically bound versus added ¹²⁵I h leptin) and as Scatchardtransformations (specific bound/free radioactivity versus specific boundradioactivity to determine the equilibrium dissociation constants[K_(d)s] (20).

For soluble receptors (sol hNR2) incubations were as above but after 1hr at 23° C., 20 μl of convavalin A-sepharose 4B beads (1/4 suspensionin 0.1 M acetate pH5) were added and incubation continued for a further30 min. Subsequenly the beads were centrifuged and processed as above.The results are shown in FIGS. 5A, 5B and 6. Human leptin binds toBa/F3/COS cells transfectd with hNR2 cDNA and to soluble hNR2.

EXAMPLE 9 EXPRESSION OF NR2 IN ANIMAL SPECIES

Genonic DNA from various sources was digested with EcoRI. This was thenblotted onto a nylon membrane (GeneScreen Plus^(SM), NEN RsearchProducts, USA). The filter was then probed using a 1.1 kb cDNA fragmentof NR2. The fragment covers the 3′ half of the first haemopoietn domainand extends to cover the whole of the second haemopoietin domain,terminating the type M fibronectin domain. The filter was prehybidisedand hybridised in 0.5M sodium phosphate, 7% w/v SDS and 1mM EDTA at 50°C. ovenight. The filter was then washed in 40 mM sodium phosphate and 1%w/v SDS at 50° C. The results are shown in FIG. 7.

EXAMPLE 10 CLONING OF THE HUMAN NR2 LOCUS

In order to obtain genomic clones of the hunaa NR2 locus, variousgenomic libraries were screened. These libraries were screened witheither oligonucleotide or CDNA probes. Oligonucleotide screeningconditions: 10×10 ¹⁶ clones were fixed to nylon filters. (Colony/PlaqueScreen™, NEN Research Products, USA). These filters were thenprehybridised in a 6xSSC buffer containing 0.2% Ficoll, 0.2% w/v bovineserum albumin, 0.2% polyvinylpyrollidine, 0.1M ATP, 50 μg/mL transferRNA, 2 mM tetra-sodium pyrophophate, 50 μg/mL herring sperm DNA and 0.1%w/v sodium azide at 37° C. for at least 2 hours. They were hybridisedovernight under the same conditions, with at least 2×10⁶ cpm/mL of³²P-labelied oligonucleotide probe. The filters were then washed in6×SSC/0.1% w/v SDS at 50-55° C. depending on the sequence of thespecific oligonucleotide (Melting Temp −10° C.).

cDNA screening conditions: 1×10⁶ clones were fixed to nylon filers.These filters were then prehybridised in a 2×SSC buffer containing 0.2%Ficoll, 0.2% w/v bovine serum albumin, 0.2% polyvinylpyrollidine, 0.1MATP, 50 μg/mL transfer RNA, 2mM tetra-sodium pyrophophate, 50 μg/mLhering sperm DNA and 0.1% w/v sodium azide at 37° C. for at least 2hours at 65° C. They were hybridised overnight under the sameconditions, with at least 2×10⁶ cpm/mL of ³²P-labelled cDNA fragment.The filters were then were then washed in 2×SSC/0.1% w/v SDS at 65° C.

EXAMPLE 11 RESTRICTION ENZYME MAPPING

The clones obtained were characterised by mapping with partialendonuclease digestion (21).

In order to determine on which figments the various exons were present,specific oligonucleotide probes were used. The various clones weredigested with a range of restriction enzymes. These were then blotted toa nylon membrane (GreenScreenPlus^(SM)), NEN Research Products, USA).Oligonucleotides derived from the cDNA sequence (and known to bespecific for a particular exon), were then hybridised to the digestedfragment. These hybridisations were done under the same conditions asmentioned above for oligonucleotides. Exons could then be mapped tospecific fragments by a positive hybridisation after overnight exposure.

Intron/exon boundary sequences were determined by sequencing across theintron/exon boundaries. Primers specific for sequence on either side ofthe boundary were used in a sequencing PCR reacion. Sequencing wasperformed on an ABI 373 sequencer using the Taq cycle sequencing kit(Applied Biosystems). These sequences were then compared to theconsensus intron/exon boundary sequence (22). The results are shown inFIG. 8 and in Table 3.

EXAMPLE 12

DETERMINATION OF AMINO ACID SEQUENCE OF hNR2

The N-terminal amino acid sequence of hNR2 was determined The resultsare shown below. The acual sequence starts at amino acid 16. Thesequence is as follows:

[SEQ ID NO:44] Asp Ser Ile Ser Ser Ser Asp Tyr Lys Asp Asp Asp          5                  10 Glu Ser Arg Tyr Pro Ile Thr Pro Trp ArgPhe Lys          15                  20 Leu Ser Xaa Met Pro Pro Xaa SerThr Tyr Asp  25                  30                  35

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the step, features, compositions and compounds referedto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

TABLE 3 Intron-Exon juctions of the human NR2 gene Exon Intron Exon size(bp) DONOR size ACCEPTOR 55  60 ATTGGG gtaagttatt [SEQ ID NO. 16]ccttttccag GTGTAT [SEQ ID NO. 29] Ig 330 AAATAG gtaagcatta [SEQ ID NO.17] tcctaacag AATTTA [SEQ ID NO. 30] SD100A 124 TGTTCT gtaagtacca [SEQID NO. 18] ttaaattcag ATGCAA [SEQ ID NO. 31] SD100A SD100B SD100B 145CACAAG gtaggttatg [SEQ ID NO. 19] tatttaacag GCTGAC [SEQ ID NO 32] Ig291 TGATTG gtaagaaaca [SEQ ID NO. 20] 0.16 ctcattacag ATGTCA [SEQ ID NO.33] SD100A′ 118 ATTGAG gtatcatagg [SEQ ID NO. 21] 2.3 tttcaaatag ATGTGA[SEQ ID NO. 34] SD100A′ 200 CTGTGG gtatgtcaag [SEQ ID NO. 22] 2.4tcttttaaag GAGCAG [SEQ ID NO. 35] SD100B′ 149 TGGAAG gtacctttta [SEQ IDNO. 23] aaatttctag TGAAGC [SEQ ID NO. 36] SD100B′ 160 TAAAAG gtctgcagag[SEQ ID NO. 24] 0.2 tattttacag ATGTAT [SEQ ID NO. 37] FnIII  83 TGGAGGgtataccaat [SEQ ID NO. 25] >7 kbp catttggcag TTCCTA [SEQ ID NO. 38]FnIII 161 CAATTC aattggtgct [SEQ ID NO. 26] tttactacag CCCCTG [SEQ IDNO. 39] FnIII′ FnIII′ Tm 106 CCAAAG gtattgtact [SEQ ID NO. 27] 1.4tcttttcag ATGATA [SEQ ID NO. 40] Cyt (Box I)  76 CATAAG gttgcttttt [SEQID NO. 28] 3 ccctttgtag AATGAA [SEQ ID NO. 41] Cyt′(NR2.2) 212 3ccttttccag AAAATG [SEQ ID NO. 42] 3′utr >1085 3 atctaaacag AGAACG [SEQID NO. 43] Consensus     AG gt^(a)/_(b)agt tc rich-cag G     

REFERENCES:

1. Du, X.X and Williams, D.A. (1994) Blood 83: 2023-2030.

2. Yang, Y.C. and Yin, T. (1992) Biofactors 4: 15-21.

3. Paul, S.R., Bennett, F., Calvetti, J.A., Kelleher, K., Wood, C.R,O'Hara, R.J.J., Leary, A.C., Sibley, B., Clark, S.C., Williams, D.A. andYang, Y.-C. (1990) Proc. Natl. Acad. Sci. USA 87: 7512.

4. Musashi, M., Clark, S.C., Sudo, T., Urdal, D.L., and Ogawa, M. (1991)Blood 78: 1448-1451.

5. Schibler, K.L, Yang, Y.C. and Christensen, R.D. (1992) Blood 80:900-3.

6. Tsuji, K., Lyman, S.D., Sudo, T., Clark, S.C., and Ogawa, M. (1992)Blood 79: 285-60.

7. Burstein, S.A., Mei, R.L., Henthorn, J., Friese, P. and turner, K.(1992)J. Cell Physiol. 153:305-12.

8. Hangoc, G., Yin, T., Cooper, S., Schendel, P., Yang, Y.C. andBroxmeyer, H.E. (1993) Blood 81: 965-72.

9. Teramura, M., Kobayashi, S., Hoshino, S., Oshimi, K. and Mizoguchi,H. (1992) Blood 79: 327-31.

10. Yonemura, Y., Kawakita, M., Masuda, T., Fujimoto, K., Kato, K. andTakatsuki, K. (1992) Exp. Hematol. 20: 1011-6.

11. Baumann, H. and Schendel, P. (1991) J. Biol. Chem. 266: 20424-7.

12. Kawashima, I., Ohsumi, J., Mita-Honjo, K., Shimoda-Takano, K.,Ishikawa, H., Sakakibara, S., Miyadai, K, and Takiguchi, Y. (1991) Febs.Lett. 283: 199-202.

13. Keller, D.C., Du, X.X. Srour, E.f., Hoffman, R and Williams, D.A.(1993) Blood 82: 1428-35.

14. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecularcloning: A Laboratory Manual Cold Spring Harbor Laboratory, Cold SpringHarbor, New York.

44 1 15 DNA Artificial Sequence Description of ArtificialSequenceSynthetic 1 rctccartcr ctcca 15 2 23 DNA Artificial SequenceDescription of Artificial SequenceSynthetic 2 taatacgact cactataggg aga23 3 19 DNA Artificial Sequence Description of ArtificialSequenceSynthetic 3 attaccctca ctaaaggga 19 4 21 DNA Artificial SequenceDescription of Artificial SequenceSynthetic 4 actagcaggg atgtagctga g 215 21 DNA Artificial Sequence Description of Artificial SequenceSynthetic5 ctcagctaca tccctgctag t 21 6 17 DNA Artificial Sequence Description ofArtificial SequenceSynthetic 6 ctgctcctat gatacct 17 7 22 DNA ArtificialSequence Description of Artificial SequenceSynthetic 7 cctcttccatcttattgctt gg 22 8 20 DNA Artificial Sequence Description of ArtificialSequenceSynthetic 8 atcggtcgtg acatacaagg 20 9 37 DNA ArtificialSequence Description of Artificial SequenceSynthetic 9 agctaagctttctagatatc caattactcc ttggaga 37 10 34 DNA Artificial SequenceDescription of Artificial SequenceSynthetic 10 agcttctaga tcaatcactctggtgttttt caat 34 11 34 DNA Artificial Sequence Description ofArtificial SequenceSynthetic 11 acgttctaga tcaaactttt atatccatga caac 3412 3909 DNA Homo sapiens unsure (27)..(68) N is a or g or c or t 12cgaattcgcg ggcgcgtcga ccgcggnccc agctcgggag acatgggggg cgttaaagct 60ctcgtggnat tatccttcag tggggstatt ggactgactt ttcttatgct gggatgtgcc 120ttagaggatt atggatttgg cagttcaccc tgaccatctt gaaaataagt tatctctgat 180ctctgtctgt atgttacttc tctcccctca ccaacggaga acaaatgtgg gcaaagtgta 240cttctctgaa gtaagatgat ttgtcaaaaa ttctgtgtgg ttttgttaca ttgggaattt 300atttatgtga taactgcgtt taacttgtca tatccaatta ctccttggag atttaagttg 360tcttgcatgc caccaaattc aacctatgac tacttccttt tgcctgctgg actctcaaag 420aatacttcaa attcgaatgg acattatgag acagctgttg aacctaagtt taattcaagt 480ggtactcact tttctaactt atccaaaaca actttccact gttgctttcg gagtgagcaa 540gatagaaact gctccttatg tgcagacaac attgaaggaa ggacatttgt ttcaacagta 600aattctttag tttttcaaca aatagatgca aactggaaca tacagtgctg gctaaaagga 660gacttaaaat tattcatctg ttatgtggag tcattattta agaatctatt caggaattat 720aactataagg tccatctttt atatgttctg cctgaagtgt tagaagattc acctctggtt 780ccccaaaaag gcagttttca gatggttcac tgcaattgca gtgttcatga atgttgtgaa 840tgtcttgtgc ctgtgccaac agccaaactc aacgacactc tccttatgtg tttgaaaatc 900acatctggtg gagtaatttt ccrgtcacct ctaatgtcag ttcagcccat aaatatggtg 960aagcctgatc caccattagg tttgcatatg gaaatcacag atgatggtaa tttaaagatt 1020tcttggtcca gcccaccatt ggtaccattt ccacttcaat atcaagtgaa atattcagag 1080aattctacaa cagttatcag agaagctgac aagattgtct cagctacatc cctgctagta 1140gacagtatac ttcctgggtc ttcgtatgag gttcaggtga ggggcaagag actggatggc 1200ccaggaatct ggagtgactg gagtactcct cgtgtcttta ccacacaaga tgtcatatac 1260tttccaccta aaattctgac aagtgttggg tctaatgttt cttttcactg catctataag 1320aaggaaaaca agattgttcc ctcaaaagag attgtttggt ggatgaattt agctgagaaa 1380attcctcaaa gccagtatga tgttgtgagt gatcatgtta gcaaagttac ttttttcaat 1440ctgaatgaaa ccaaacctcg aggaaagttt acctatgatg cagtgtactg ctgcaatgaa 1500catgaatgcc atcatcgcta tgctgaatta tatgtgattg atgtcaatat caatatctca 1560tgtgaaactg atgggtactt aactaaaatg acttgcagat ggtcaaccag tacaatccag 1620tcacttgcgg aaagcacttt gcaattgagg tatcatagga gcagccttta ctgttctgat 1680attccatcta ttcatcccat atctgagccc aaagattgct atttgcagag tgatggtttt 1740tatgaatgca ttttccagcc aatcttccta ttatctggct acacaatgtg gattaggatc 1800aatcactctc taggttcact tgactctcca ccaacatgtg tccttcctga ttctgtggtg 1860aagccactgc ctccatccag tgtgaaagca gaaattacta taaacattgg attattgaaa 1920atatcttggg aaaagccagt ctttccagag aataaccttc aattccagat tcgctatggt 1980ttaagtggaa aagaagtaca atggaagatg tatgaggttt atgatccaaa accaaaatct 2040gtcagtctcc cagttccaga cttgtgtgca gtctatgctg ttcaggtggc gtttaagagg 2100ctagatggac tgggatattg gagtaattgg agcaatccag cctacacagt tgtcatggat 2160ataaaagttc ctatgagagg acctgaattt tggagaataa ttaatggaga tactatgaaa 2220aaggagaaaa atgtcacttt actttggaag cccctgatga aaaatgactc attgtgcagt 2280gttcagagat atgtgataaa ccatcatact tcctscaatg gaacatggtc agaagatgtg 2340ggaaatcaca cgaaattcac tttcctgtgg acagagcaag cacatactgt tacggttctg 2400gccatcaatt caattggtgc ttctgttgca aattttaatt taaccttttc atggcctatg 2460agcaaagtaa atatcgtgca gtcactcagt gcttatcctt taaacagcag ttgtgtgatt 2520gtttcctgga tactatcacc cagtgattac aagctaatgt attttattat tgagtggaaa 2580aatcttaatg aagatggtga aataaaatgg cttagaatct cttcatctgt taagaagtat 2640tatatccatg atcattttat ccccattgag aagtaccagt tcagtcttta cccaatattt 2700atggaaggag tgggaaaacc aaagataatt aatagtttca ctcaagatga tattgaaaaa 2760caccagagtg atgcaggttt atatgtaatt gtgccagtaa ttatttcctc ttccatctta 2820ttgcttggaa cattattaat atcacaccaa agaatgaaaa agctattttg ggaagatgtt 2880ccgaacccca agaattgttc ctgggcacaa ggacttaatt ttcagaagag aacggacatt 2940ctttgaagtc taatcatgat cactacagat gaacccaatg tgccaacttc ccaacagtct 3000atagagtatt agaagatttt tacattttga agaaggggag caaatctaaa aaaaattcag 3060ttgaacttct gagagttaac atatggtgga ttatgttgat ttagaactta aaatagatgt 3120catttaaacc caagttttac atctaaactc aggtcaaacc tacacactaa ttaaaagttt 3180agtagatttc aaattttcat cataagtact aaagaccgaa aactaaacag tataaggacc 3240agtattttgt aattctttta ataccgacaa cgacagtaat gtatagataa tttacagtag 3300tttatacatc atctgttagg acattaatcc acttgagatt ttgacgttgt agactgttta 3360tcgaaatttt tatgttacta atattcatac cttagtcact tttataaatc aaacataaaa 3420atacaggttt gaaaaggtaa aatctaagga aatatctgtg cagtcggatt tttagtcgga 3480taagcccaca agaaaactta tagaggaccg taaaaacata gattgaaaca agttagaccc 3540ttaaagtcaa aagttatagg aacttttacc gaattcacta ttgaaggcaa agtcaatttt 3600ccttcgggct tcaacacaaa cacgacgggt gtcctgtcac cctcaatgtc aagtatagtc 3660ctactgggat gtatgggtcc agtctaactg ccctggtctt cccttgtagc tgaagattac 3720aggtgcgaaa gaacaaatta atactggatt tagattaaat gaaggtgact tggtaggttc 3780tggagaccgt ccgtcccttt acccgtcact asgttttttc cctctgagaa acctcgaaaa 3840tacttatcaa gtaccactcc tgtcttgaaa agatgaaagt ctgtctgacg aacgatcaaa 3900atacttaag 3909 13 896 PRT Homo sapiens UNSURE (223) Xaa is unknown orother. 13 Met Ile Cys Gly Lys Phe Cys Val Val Leu Leu His Trp Gln PheIle 1 5 10 15 Tyr Val Ile Thr Ala Phe Asn Leu Ser Tyr Pro Ile Thr ProTrp Arg 20 25 30 Phe Lys Leu Ser Cys Met Pro Pro Asn Ser Thr Thr Asn TyrPhe Leu 35 40 45 Leu Pro Ala Gly Leu Ser Lys Asn Thr Ser Asn Ser Asn GlyHis Tyr 50 55 60 Glu Thr Ala Val Glu Pro Lys Phe Asn Ser Ser Gly Thr HisPhe Ser 65 70 75 80 Asn Leu Ser Lys Thr Thr Phe His Cys Cys Phe Arg SerGlu Gln Asp 85 90 95 Arg Asn Cys Ser Leu Cys Ala Asp Asn Ile Glu Gly ArgThr Phe Val 100 105 110 Ser Thr Val Asn Ser Leu Val Phe Gln Gln Ile AspAla Asn Trp Asn 115 120 125 Ile Gln Cys Trp Leu Lys Gly Asp Leu Lys LeuPhe Ile Cys Tyr Val 130 135 140 Glu Ser Leu Phe Lys Asn Leu Phe Arg AsnTyr Asn Tyr Lys Val His 145 150 155 160 Leu Leu Tyr Val Leu Pro Glu ValLeu Glu Asp Ser Pro Leu Val Pro 165 170 175 Gln Lys Gly Ser Phe Gln MetVal His Cys Asn Cys Ser Val His Glu 180 185 190 Cys Cys Glu Cys Leu ValPro Val Pro Thr Ala Lys Leu Asn Asp Thr 195 200 205 Leu Leu Met Cys LeuLys Ile Thr Ser Gly Gly Val Ile Phe Xaa Ser 210 215 220 Pro Leu Met SerVal Gln Pro Ile Asn Met Val Lys Pro Asp Pro Pro 225 230 235 240 Leu GlyLeu His Met Glu Ile Thr Asp Asp Gly Asn Leu Lys Ile Ser 245 250 255 TrpSer Ser Pro Pro Leu Val Pro Phe Pro Leu Gln Tyr Gln Val Lys 260 265 270Tyr Ser Glu Asn Ser Thr Thr Val Ile Arg Glu Ala Asp Lys Ile Val 275 280285 Ser Ala Thr Ser Leu Leu Val Asp Ser Ile Leu Pro Gly Ser Ser Tyr 290295 300 Glu Val Gln Val Arg Gly Lys Arg Leu Asp Gly Pro Gly Ile Trp Ser305 310 315 320 Asp Trp Ser Thr Pro Arg Val Phe Thr Thr Gln Asp Val IleTyr Phe 325 330 335 Pro Pro Lys Ile Leu Thr Ser Val Gly Ser Asn Val SerPhe His Cys 340 345 350 Ile Tyr Lys Lys Glu Asn Lys Ile Val Pro Ser LysGlu Ile Val Trp 355 360 365 Trp His Asn Leu Ala Glu Leu Ile Pro Gln SerGln Tyr Asp Val Val 370 375 380 Ser Asp His Val Ser Lys Val Thr Phe PheAsn Leu Asn Glu Thr Lys 385 390 395 400 Pro Arg Gly Leu Phe Thr Tyr AspAla Val Tyr Cys Cys Asn Glu His 405 410 415 Gly Cys His His Arg Tyr AlaGly Leu Tyr Val Ile Asn Val Asn Ile 420 425 430 Asn Ile Ser Cys Gln ThrAsn Gly Tyr Leu Thr Lys Met Thr Cys Arg 435 440 445 Trp Ser Thr Ser ThrIle Gln Ser Leu Ala Glu Ser Thr Leu Glu Leu 450 455 460 Arg Tyr His ArgSer Ser Leu Tyr Cys Ser Asn Ile Pro Ser Ile His 465 470 475 480 Pro IleSer Glu Pro Lys Asn Cys Tyr Leu Gln Ser Asn Gly Phe Tyr 485 490 495 GlnCys Ile Pro Gln Pro Ile Phe Leu Leu Ser Gly Tyr Thr Met Trp 500 505 510Ile Arg Ile Asn His Ser Leu Gly Ser Leu Asn Ser Pro Pro Thr Cys 515 520525 Val Leu Pro Asp Ser Val Val Lys Pro Leu Pro Pro Ser Ser Val Lys 530535 540 Ala Glu Ile Thr Ile Asn Ile Gly Leu Leu Lys Ile Ser Trp Glu Lys545 550 555 560 Pro Val Phe Pro Glu Asn Asn Leu Gln Phe Gln Ile Arg ThrGly Leu 565 570 575 Ser Gly Lys Glu Val Gln Trp Lys Met Tyr Glu Val ThrAsn Pro Lys 580 585 590 Pro Lys Ser Val Ser Leu Pro Val Pro Asp Leu CysAla Val Tyr Ala 595 600 605 Val Gln Val Arg Phe Lys Arg Leu Asp Gly LeuGly Tyr Trp Ser Asn 610 615 620 Trp Ser Asn Pro Ala Tyr Thr Val Val MetAsp Ile Lys Val Pro Met 625 630 635 640 Arg Gly Pro Glu Phe Trp Arg IleIle Asn Gly Asp Thr Met Lys Lys 645 650 655 Glu Lys Asn Val Tyr Leu LeuTrp Lys Pro Leu Met Lys Asn Asp Ser 660 665 670 Leu Cys Ser Val Gln ArgTyr Val Ile Asn His His Thr Ser Xaa Asn 675 680 685 Gly Thr Trp Ser GluAsn Val Gly Asn His Thr Lys Phe Thr Phe Leu 690 695 700 Trp Thr Glu GlnAla His Thr Val Thr Val Leu Ala Ile Asn Ser Ile 705 710 715 720 Gly AlaSer Val Ala Asn Phe Asn Leu Thr Phe Ser Trp Pro Met Ser 725 730 735 LysVal Asn Ile Val Gln Ser Leu Ser Ala Tyr Pro Leu Asn Ser Ser 740 745 750Cys Val Ile Val Ser Trp Ile Leu Ser Pro Ser Asp Val Lys Leu Met 755 760765 Tyr Pro Ile Ile Glu Trp Lys Asn Leu Asn Glu Asp Gly Glu Ile Lys 770775 780 Trp Leu Arg Ile Ser Ser Ser Val Lys Lys Tyr Tyr Ile His Asp His785 790 795 800 Phe Ile Pro Ile Glu Lys Tyr Gln Phe Ser Leu Tyr Pro IlePhe Met 805 810 815 Glu Gly Val Gly Lys Pro Lys Ile Ile Asn Ser Phe ThrGln Asn Asn 820 825 830 Ile Glu Lys His Gln Ser Asp Ala Gly Leu Tyr ValIle Val Pro Val 835 840 845 Ile Ile Ser Ser Ser Ile Leu Leu Leu Gly ThrLeu Leu Ile Ser His 850 855 860 Gln Arg Met Lys Lys Leu Phe Trp Glu AspVal Pro Asn Pro Lys Asn 865 870 875 880 Cys Ser Trp Ala Gln Gly Leu AsnPhe Gln Lys Arg Thr Asn Ile Leu 885 890 895 14 30 PRT ArtificialSequence Description of Artificial SequenceSynthetic 14 Met Val Leu AlaSer Ser Thr Thr Ser Ile His Thr Met Leu Leu Leu 1 5 10 15 Leu Leu MetLeu Pro His Leu Gly Leu Gly Ala Ser Ile Ser 20 25 30 15 8 PRT ArtificialSequence Description of Artificial SequenceSynthetic 15 Ala Thr Leu AlaAla Ala Ala Leu 1 5 16 16 DNA Artificial Sequence Description ofArtificial SequenceSynthetic 16 attggggtaa gttatt 16 17 16 DNAArtificial Sequence Description of Artificial SequenceSynthetic 17aaataggtaa gcatta 16 18 16 DNA Artificial Sequence Description ofArtificial SequenceSynthetic 18 tgttctgtaa gtacca 16 19 16 DNAArtificial Sequence Description of Artificial SequenceSynthetic 19cacaaggtag gttatg 16 20 16 DNA Artificial Sequence Description ofArtificial SequenceSynthetic 20 tgattggtaa gaaaca 16 21 16 DNAArtificial Sequence Description of Artificial SequenceSynthetic 21attgaggtat catagg 16 22 16 DNA Artificial Sequence Description ofArtificial SequenceSynthetic 22 ctgtgggtat gtcaag 16 23 16 DNAArtificial Sequence Description of Artificial SequenceSynthetic 23tggaaggtac ctttta 16 24 16 DNA Artificial Sequence Description ofArtificial SequenceSynthetic 24 taaaaggtct gcagag 16 25 16 DNAArtificial Sequence Description of Artificial SequenceSynthetic 25tggagggtat nccaat 16 26 16 DNA Artificial Sequence Description ofArtificial SequenceSynthetic 26 caattcaatt ggtgct 16 27 16 DNAArtificial Sequence Description of Artificial SequenceSynthetic 27ccaaaggtat tgtact 16 28 16 DNA Artificial Sequence Description ofArtificial SequenceSynthetic 28 cataaggttg cttttt 16 29 16 DNAArtificial Sequence Description of Artificial SequenceSynthetic 29ccttttccag gtgtat 16 30 15 DNA Artificial Sequence Description ofArtificial SequenceSynthetic 30 tcctaacaga attta 15 31 16 DNA ArtificialSequence Description of Artificial SequenceSynthetic 31 ttaaattcagatgcaa 16 32 16 DNA Artificial Sequence Description of ArtificialSequenceSynthetic 32 tatttaacag gctgac 16 33 16 DNA Artificial SequenceDescription of Artificial SequenceSynthetic 33 ctcattacag atgtca 16 3416 DNA Artificial Sequence Description of Artificial SequenceSynthetic34 tttcaaatag atgtga 16 35 16 DNA Artificial Sequence Description ofArtificial SequenceSynthetic 35 tcttttaaag gagcag 16 36 16 DNAArtificial Sequence Description of Artificial SequenceSynthetic 36aaatttctag tgaagc 16 37 16 DNA Artificial Sequence Description ofArtificial SequenceSynthetic 37 tattttacag atgtat 16 38 16 DNAArtificial Sequence Description of Artificial SequenceSynthetic 38catttggcag ttccta 16 39 16 DNA Artificial Sequence Description ofArtificial SequenceSynthetic 39 tttactacag cccctg 16 40 16 DNAArtificial Sequence Description of Artificial SequenceSynthetic 40tctttttcag atgata 16 41 16 DNA Artificial Sequence Description ofArtificial SequenceSynthetic 41 ccctttgtag aatgaa 16 42 16 DNAArtificial Sequence Description of Artificial SequenceSynthetic 42ccttttccag aaaatg 16 43 16 DNA Artificial Sequence Description ofArtificial SequenceSynthetic 43 atctaaacag agaacg 16 44 32 PRTArtificial Sequence UNSURE (27) Xaa is unknown or other. 44 Ala Ser IleSer Ser Ser Ala Thr Leu Ala Ala Ala Gly Ser Ala Thr 1 5 10 15 Pro IleThr Pro Thr Ala Pro Leu Leu Ser Xaa Met Pro Pro Xaa Ser 20 25 30

What is claimed is:
 1. An isolated nucleic acid molecule encoding athaemopoietin receptor, wherein said haemopoietin receptor comprises theamino acid sequence as set forth in SEQ ID NO:13.
 2. An isolated nucleicacid molecule comprising the nucleotide sequence set forth in SEQ IDNO:12.
 3. An isolated nucleic acid molecule, wherein said nucleic acidmolecule encodes a portion of haemopoietin receptor which comprises theamino acid sequence as set forth in SEQ ID NO: 13, and wherein saidportion of the haemopoietin receptor binds leptin and comprises theamino acid sequence of Tyr26 to Asp839 of SEQ ID NO:
 13. 4. A vectorcomprising the nucleic acid molecule according to claim 1, 2 or
 3. 5. Ahost cell comprising the vector according to claim
 4. 6. A host cellcomprising the nucleic acid molecule according to claim 1, 2 or 3.